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Title Studies on Genetic Diversity of Spotted Fever Group Rickettsiae in Ixodid Ticks in Japan
Author(s) THU, May June
Citation 北海道大学. 博士(獣医学) 甲第13506号
Issue Date 2019-03-25
DOI 10.14943/doctoral.k13506
Doc URL http://hdl.handle.net/2115/74789
Type theses (doctoral)
File Information May_June_THU.pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
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Studies on Genetic Diversity of Spotted
Fever Group Rickettsiae in Ixodid Ticks
in Japan
(日本産マダニが保有する紅斑熱群リケッチアの遺伝的多様性に関する研究)
May June THU
ii
Notes
The contents of chapter I have been submitted in Scientific Reports.
Thu MJ, Qiu Y, Matsuno K, Kajihara M, Mori-Kajihara A, Omori R,
Monma N, Chiba K, Seto J, Gokuden M, Andoh M, Oosako H,
Katakura K, Takada A, Sugimoto C, Isoda N, Nakao R. Diversity of
spotted fever group rickettsiae and their association with host ticks in
Japan. Sci Rep In press.
The contents of chapter II have been submitted in Vector-Borne and
Zoonotic Diseases.
Thu MJ, Qiu Y, Kataoka-Nakamura C, Sugimoto C, Katakura K,
Isoda N, Nakao R. Isolation of Rickettsia, Rickettsiella, and
Spiroplasma from questing ticks in Japan using arthropod cells. Vector
Borne Zoonotic Dis In press.
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TABLE OF CONTENTS
Page
TABLE OF CONTENTS i
ABBREVIATIONS iii
GENERAL INTRODUCTION 1
CHAPTER I Diversity of spotted fever group rickettsiae and their
association with host ticks in Japan
1 Introduction 8
2 Materials and methods 9
2.1 Sample collection
2.2 Tick species identification
2.3 DNA extraction
2.4 Real-time PCR
2.5 Conventional PCR
2.6 Sequencing
2.7 Phylogenetic analysis
3 Results 13
3.1 Detection of SFG rickettsiae by real-time PCR
3.2 gltA sequence genotyping
3.3 Multiple genes sequencing
3.4 Species identification of SFG rickettsiae
4 Discussion 16
5 Summary 22
ii
CHAPTER II Isolation of Rickettsia, Rickettsiella, and Spiroplasma
from questing ticks in Japan using arthropod cells
1 Introduction 38
2 Materials and methods 40
2.1 Tick samples
2.2 Maintenance of cell lines
2.3 Co-culture with tick homogenates
2.4 Conventional PCR
2.5 Real-time PCR
2.6 Sequencing and data analysis
3 Results 44
3.1 Real-time PCR
3.2 Isolation results
3.3 Rickettsia isolation
3.4 Rickettsiella isolation
3.5 Spiroplasma isolation
4 Discussion 47
5 Summary 51
GENERAL CONCLUSIONS 64
ACKNOWLEDGEMENTS 66
REFERENCES 69
JAPANESE ABSTRACT 84
ABSTRACT 86
iii
ABBREVIATIONS
16S rRNA 16S ribosomal RNA
% percentage
µL microliter
AG ancestral group
bp base pair
C degree Celsius
DDBJ DNA data bank of Japan
DNA deoxyribonucleic acid
DMEM Dulbecco’s Modified Eagle Medium
JSF Japanese spotted fever
gltA citrate synthase gene
htrA 17-kDa common antigen gene
kDa kilo Dalton
min minute
No. number
nM nanomolar
ompA outer membrane protein A gene
ompB outer membrane protein B gene
PBS phosphate-buffered saline
PCR polymerase chain reaction
RNA ribonucleic acid
sca surface cell antigen gene
SFG spotted fever group
sec second
TG typhus group
TRG transitional group
USA The United States of America
V voltage
1
GENERAL INTRODUCTION
The term Rickettsiae represents a group of microorganisms in the genus
Rickettsia within the family Rickettsiaceae in the order Rickettsiales that belongs
to the class Alphaproteobacteria (Raoult and Roux, 1997). The genus Rickettsia was
named after Howard Taylor Ricketts, who died from murine typhus during the study
of Rickettsiae. Rickettsiosis is one of the oldest vector-borne infectious diseases as
it was evident as early as the end of 16th century (Raoult and Roux, 1997).
Originally, Rickettsia was considered as a virus or an unknown organism because
of the small size and variable morphology, and it was not easy to identify them
under the light microscope (Wolbach, 1919; Parola et al., 2005). Rickettsia is a short
rod-shaped with 0.3 to 0.5 × 0.8 to 2.0 µm in size and has a typical Gram-negative
cell wall on the outside of a thin cytoplasmic membrane. Within the host cell,
Rickettsia is surrounded by a slime layer or little capsular material built up by
lipopolysaccharide, characterized as a halo around the bacterium when imaged by
electron microscopy (Winkler, 1990; Parola et al., 2005; Raoult et al., 2005).
The members of the genus Rickettsia are divided into four main groups: the
spotted fever group (SFG), typhus group (TG), transitional group (TRG), and
ancestral group (AG) (Gillespie et al., 2007). SFG and AG rickettsiae are mainly
associated with ticks, while TG and TRG rickettsiae are associated with other
arthropods such as lice, fleas, and mites. TG is composed of Rickettsia typhi and
Rickettsia prowazekii, while TRG is composed of Rickettsia akari, Rickettsia
australis, and Rickettsia felis. Among the tick-borne rickettsiae, AG includes
Rickettsia bellii and Rickettsia canadensis. More than 25 species of tick-borne
rickettsiae that have been validated so far belong to SFG. Furthermore, the members
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of SFG rickettsiae have been increasing as many new species have been proposed
recently (Karpathy et al., 2016; Abdad et al., 2017; Dall'Agnol et al., 2017; Lee et
al., 2017; Moreira-Soto et al., 2017).
More than a century ago, the taxonomy of Rickettsia has been based on the
comparative studies of morphology, antigenic and physiological characters
(Fournier and Raoult, 2009). However, the studies of morphological and
physiological variation are inadequate to classify all the members of the genus
Rickettsia. Phylogenetic analysis inferred from the sequences of the 16S ribosomal
RNA (rRNA) gene enabled the reclassification of rickettsiae (Weisburg et al., 1991;
Raoult and Roux, 1997). Additionally, the outer membrane protein family genes
such as outer membrane protein A gene (ompA), outer membrane protein B gene
(ompB) and surface cell antigen-4 gene (sca4) have been used to further refine the
classification of rickettsiae on the basis of their sequence similarities between
rickettsial species (Blanc et al., 2005; Fournier and Raoult, 2009; Phan et al., 2011).
The ompA is specific to SFG rickettsiae but not found in all the members of genus
Rickettsia (Fournier et al., 2003), while the ompB is retained in both SFG and TG
group rickettsiae and has functions associated with invasion and adhesion to the
host cells (Roux and Raoult, 2000; Chan et al., 2010). Moreover, the citrate
synthase gene (gltA) encoding an enzyme essential in the central metabolic
pathways of the rickettsiae and 17-kDa common antigen gene (htrA) are useful to
characterise species of SFG rickettsiae because their sequences are relatively
conserved within the same species and sequence data of representative rickettsial
species are available in the database (Fournier et al., 2003; Labruna et al., 2004).
In Asia, several pathogenic SFG rickettsiae are known to exist. For
instance, R. sibirica, the etiological agent of Siberian tick typhus was reported from
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Russia, Mongolia, and China (Mediannikov et al., 2004; Shpynov et al., 2006). Thai
tick typhus caused by R. honei was reported from Thai-Myanmar border and Nepal
(Parola et al., 2003; Murphy et al., 2011). Indian tick typhus caused by R. conorii
was reported from India and Mongolia (Parola et al., 2001; Rolain et al., 2003).
Candidatus Rickettsia kellyi, R. raoultii and R. monacensis have been associated
with unknown spotted fever in India, China and South Korea, respectively (Rolain
et al, 2006; Jia et al., 2014; Kim et al., 2017). R. felis, the causative agent of flea-
borne spotted fever has been reported in Vietnam and Laos (Phongmany et al.,
2006).
So far four pathogenic SFG rickettsiae, including R. japonica, Rickettsia
heilongjiangensis, Rickettsia helvetica, and Rickettsia tamurae are known to be
endemic in Japan (Mahara, 1997; Noji et al., 2005; Ando et al., 2010; Imaoka et al.,
2011). Human infected with Rickettsia organisms typically has high fever,
headache, and skin eruption. In general, infected humans develop febrile reaction
for 1 to 2 weeks and the period can vary depending on the virulence of the
organisms (Mahara, 1997). In severe cases, swelling of liver and spleen,
cardiomegaly and nervous signs may occur. Death may be due to pericarditis or the
result of cytolytic toxin (Mahara, 1997; Imaoka et al., 2011).
Even though the genetic detection and classification of Rickettsia could be
achieved by PCR-based molecular techniques, the pathogenicity to vertebrate
animals is not well understood in most of the rickettsial species, especially those
newly found in ticks. The lines of evidence showed some of the Rickettsia without
any known vertebrate pathogenicity have symbiotic association with their arthropod
hosts (Fournier and Raoult, 2009). In some cases, rickettsiae play roles as
endosymbionts presumably with some benefit to host ticks, but this is not always
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true (Perlman et al., 2006). In the natural environment, ticks become infected with
rickettsiae when they feed on hosts carrying the bacteria or vertically through
transovarial and transstadial transmission (Raoult and Roux, 1997) (Figure 1). The
presence of Rickettsiae in the eggs of tick, indicating potential of vertical
transmission, is usually an indication of symbiotic interactions between Rickettsia
and ticks. In general, horizontal transmission of bacteria across different host
lineages tends to exacerbate the virulence of bacteria to their hosts, whereas vertical
transmission through host generations tends to attenuate their virulence, potentially
leading to commensalism and ultimately to mutualism (Dale and Moran, 2006). At
present, the nature of endosymbionts of ticks including rickettsiae and their
relationships to ticks remain poorly understood.
Ticks are obligate blood-sucking arthropods found in tropical and
temperate regions of the world (Parola et al., 2005). In Japan, tick fauna is
composed of seven genera: Argas, Ornithodoros, Amblyomma, Dermacentor,
Haemaphysalis, Ixodes and Rhipicephalus, and 47 different tick species are
reported so far (Nakao et al., 1992; Nakao and Ito, 2014; Kwak, 2018). Out of 47
tick species, 20 species are reported to bite humans (Yamauchi et al., 2010;
Seishima et al., 2000; Nakamura-Uchiyama et al., 2000; Ando et al., 2010).
According to the Japanese Infectious Agents Surveillance Report, rickettsiosis has
counted the highest number of cases among all tick-borne bacterial diseases in
Japan (Yamaji et al., 2018). Despite the availability of effective antibiotic treatment,
fatal cases of rickettsiosis have continued to be reported annually. The mortality
rate of some rickettsial diseases such as Japanese spotted fever (JSF) is still
indicating 0.91% (National Institute of Infectious Diseases 2017), emphasizing a
public health importance of Rickettsia. However, little is known about the
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prevalence of Rickettsia species in hard ticks and bacterial endosymbionts of
questing ticks in Japan. It is highly possible that several unrecognised Rickettsia
species are expected to exist in Japan. The data obtained from this study will
provide information on the genetic diversity of rickettsial organisms which may
include previously unrecognised rickettsial agents as well as the geographical
distribution of known rickettsial pathogens in Japan.
In the first chapter of this thesis, a national wide cross-sectional survey of
ixodid ticks in Japan was conducted to evaluate the relationship between SFG
rickettsiae and their vector ticks. In addition, the multiple gene analysis was
performed to provide the detail comprehensive taxonomy and phylogeny of
Rickettsia. In the second chapter, isolation of SFG rickettsiae and tick
endosymbionts was attempted from ticks using arthropods cells. Further genetic
classification of Rickettsia species and tick symbionts was conducted.
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Figure 1. Life cycle of ixodid ticks and transmission of rickettsiae.
Yellow arrows indicate transstadial transmission of rickettsiae and orange arrow
indicates transovarial transmission of rickettsiae. (1) Tick eggs hatch into larvae.
(2) Larvae feed on small animals. (3) Larvae moult into nymphs. (4) Nymphs feed
on animals and humans. (5) Nymphs moult into adult ticks. (6) Oviposition by
engorged female. (7) Adult ticks feed on animals or bite humans.
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CHAPTER I
Diversity of spotted fever group rickettsiae and their association with host
ticks in Japan
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1 INTRODUCTION
In Japan, Rickettsia japonica was the first Spotted fever group (SFG)
Rickettsia discovered in 1984 as the causative agent of Japanese spotted fever (JSF)
(Uchida et al., 1986; Mahara, 1997). Since then, several other SFG rickettsiae,
namely Rickettsia heilongjiangensis, Rickettsia helvetica, and Rickettsia tamurae
have been recognised as etiological agents of human diseases (Noji et al., 2005;
Ando et al., 2010; Imaoka et al., 2011). SFG rickettsiae with unknown
pathogenicity, such as Rickettsia asiatica and Candidatus R. tarsevichiae, have also
been reported (Fujita et al., 2006; Inokuma et al., 2007). In addition, several studies
conducted in Japan have documented the presence of other Rickettsia
species/genotypes in animals and questing ticks (Baba et al., 2013; Gaowa et al.,
2013; Someya et al., 2015). However, in most cases, only single or a limited number
of genes have been analysed, making it difficult to generate an overview of the
genetic diversity of SFG rickettsiae, since multiple gene sequencing are
recommended in the classification of rickettsial isolates (Fournier et al., 2003).
The relationship between SFG rickettsiae and their vector tick species has
been studied previously. It is evident that some SFG rickettsiae, such as Rickettsia
rickettsii, are associated with several different tick species in different genera, while
others, such as R. conorii, are linked to specific tick species (Socolovschi et al.,
2009). In Japan, R. japonica is considered to be in the former group since it has
been recorded from wide range of tick species including Dermacentor taiwanensis,
Haemaphysalis hystricis, Haemaphysalis cornigera, Haemaphysalis longicornis,
Haemaphysalis flava, Haemaphysalis formosensis, Haemaphysalis megaspinosa,
and Ixodes ovatus (Ando and Fujita, 2013). On the other hand, vector tick species
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of other rickettsiae, such as R. asiatica and R. heilongjiangensis, which are
respectively transmitted by I. ovatus and H. concinna, seem to be limited (Fujita et
al., 2006; Ando et al., 2010).
The aim of this chapter was to understand the overall diversity of SFG
rickettsiae and their vector tick species in Japan. By collecting questing ticks at
more than 100 different sampling sites across Japan, a nationwide cross-sectional
study for SFG rickettsiae was conducted. The samples included 19 different tick
species covering most of the commonly found species in Japan. Our results indicate
that there exist more SFG rickettsiae genotypes than previously known. The
information on the relationship between SFG rickettsiae and vector ticks is useful
for further characterisation of each rickettsiae member in more detail.
2 MATERIALS AND METHODS
2.1 Sample collection
Ticks were collected by flagging a flannel cloth over the vegetation during
the period of tick activity (between April 2013 and March 2016) at 114 different
sampling sites in 12 different prefectures. The sampling sites were categorised into
geographical blocks: Hokkaido (Hokkaido prefecture), Tohoku (Yamagata and
Fukushima prefectures), Chubu (Nagano and Shizuoka prefectures), Kansai (Mie,
Nara, and Wakayama prefectures), Kyushu (Kumamoto, Miyazaki, and Kagoshima
prefectures), and Okinawa (Okinawa prefecture) (Figure I-1). All field-collected
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ticks were transferred to small petri dishes and preserved in an incubator at 16°C
until use.
2.2 Tick species identification
Tick species were identified morphologically using standard keys under a
stereomicroscope (Yamaguti et al., 1971; Nakao et al., 1992). When more than 10
ticks with the same species and stage/sex were collected from the same sampling
sites, a maximum of 10 individual ticks were analysed per species, stage/sex and
site. A total of 2,189 individuals (103 nymphs and 2,086 adults) in four genera were
examined in this study. These included one species in the genus Amblyomma (A.
testudinarium, n = 85), one species in the genus Dermacentor (D. taiwainensis, n =
12), 10 species in the genus Haemaphysalis (H. concinna, n = 7; H. cornigera, n =
1; H. flava, n = 128; H. formosensis, n = 253; H. japonica, n = 78; H. hystricis, n =
64; H. kitaokai, n = 74; H. longicornis, n = 86; H. megaspinosa, n = 201; and H.
yeni, n = 1) and 7 species in the genus Ixodes (I. monospinosus, n = 58; I.
nipponensis, n = 5; I. ovatus, n = 652; I. pavlovskyi, n = 33; I. persulcatus, n = 446;
I. tanuki, n = 2; and I. turdus, n = 3). Out of 2,189 ticks, 975, 1,111, and 103 were
male, female, and nymph, respectively.
2.3 DNA extraction
Ticks were individually washed with 70% ethanol followed by washing
with sterile PBS twice, then homogenised in 100 μL of high glucose Dulbecco’s
Modified Eagle Medium (DMEM, Gibco, Life Technologies) by using Micro
Smash MS100 (TOMY, Tokyo, Japan) for 30 sec at 3,000 rpm as described
previously (Nakao et al., 2011). DNA was extracted from 50 μL of the tick
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homogenate using a blackPREP Tick DNA/RNA Kit (Analytikjena, Germany)
according to the manufacturer’s instructions, while the other half was kept at -80°C
for future bacterial isolation.
2.4 Real-time PCR
All samples were first screened for citrate synthase gene (gltA) using real-
time PCR to detect SFG and TG rickettsiae as described previously (Stenos et al.,
2005). The primers and probes used are shown in Table I-1. Reactions were
performed in a 20 μL of reaction mixture containing 10 μL of THUNDERBIRD
Probe qPCR Mix (Toyobo, Osaka, Japan), 300 nM of each primer, 200 nM of probe,
5.0 μL of template DNA, and distilled water. The reaction was carried out in a
C1000 Thermal Cycler with a CFX96 Real-Time PCR Detection System (Bio-Rad
Laboratories, Hercules, CA) at conditions of 50°C for 3 min, 95°C for 1 min, and
40 cycles of 95°C for 15 sec and 60°C for 1 min. Each run included a negative
control and serially diluted plasmid standards (106, 104, and 102 copies/reaction) as
described previously (Nakao et al., 2013).
2.5 Conventional PCR
All the samples that were positive for gltA by real-time PCR were further
characterised by conventional PCR targeting an approximately 580 bp sequence of
the gltA gene using the primers gltA–Fc and gltA–Rc (Table I-1) (Gaowa et al.,
2013). The PCR was carried out in a 25 μL reaction mixture containing 12.5 μL of
2×KAPA blood PCR Kit (KAPA Bio systems, USA), 200 nM of each primer, 2.0
μL of DNA template, and sterile water. The reactions were performed at 95°C for
5 min; followed by 45 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 40
12
sec; and 72°C for 5 min. PCR products were electrophoresed at 100 V in a 1.2%
agarose gel for 25 min. DNA from the R. japonica YH strain and sterile water were
included in each PCR run as positive and negative controls, respectively.
For the selected samples from each gltA genotype (n = 57), additional PCR
assays were conducted based on five genes: the 190-kDa outer membrane protein
A (ompA), 120-kDa outer membrane protein B (ompB), surface cell antigen-4
(sca4), 17-kDa common antigen (htrA), and 16S rRNA. The primer sets used for
each assay are shown in Table I-1 (Regnery et al., 1991; Roux and Raoult, 2000;
Sekeyova et al., 2001; Labruna et al., 2004; Anstead and Chilton, 2013). PCR
conditions were the same as mentioned above except for the annealing temperature
(48°C for ompA and ompB PCRs, 52°C for 16S rRNA and htrA PCRs, and 50°C
for sca4 PCR).
2.6 Sequencing
The amplified PCR products were purified using a Wizard® SV Gel and
PCR Clean-Up System Kit (Promega, USA). Sanger sequencing was conducted
using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied
Biosystems, Foster City, CA) and an ABI Prism 3130xl Genetic Analyzer Kit
(Applied Biosystems) according to the manufacturers’ instructions. The sequences
data were assembled using ATGC software version 6.0.4 (GENETYX, Tokyo,
Japan). The sequences obtained were submitted to the DNA Data Bank of Japan
(DDBJ) (http://www.ddbj.nig.ac.jp) under accession numbers (gltA: LC379427-
LC379443; ompA: LC379461-LC379465; ompB: LC379466-LC379476; htrA:
LC379444-LC379460; sca4: LC379477-LC379482; 16S rRNA: LC379483-
LC379494).
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2.7 Phylogenetic analysis
The nucleotide sequences obtained were aligned with representative
sequences of known rickettsial species available on GenBank using ClustalW 1.6
as implemented in MEGA 7 (Kumar et al., 2016). After manual modification of the
alignments, phylogenetic trees were constructed using the maximum likelihood
method using Kimura 2-parameter with bootstrap tests of 1,000 replicates via
MEGA. R. bellii was included as an outgroup for the bases of the trees for gltA,
ompB, and htrA, while R. typhi, R. akari, and Ehrlichia chaffeensis were used as
outgroups for sca4, ompA, 16S rRNA, respectively. In order to generate a
phylogenetic tree of tick species that was positive for Rickettsia spp., partial
nucleotide sequences of mitochondrial 16S rRNA gene obtained from GenBank
were used.
3 RESULTS
3.1 Detection of SFG rickettsiae by real-time PCR for gltA
Out of 2,189 ticks, 373 (17.0 %) samples were positive for Rickettsia spp.
by gltA real-time PCR (Table I-2). Among the 19 different tick species, seven tick
species, namely D. taiwainensis, H. concinna, H. cornigera, H. yeni, I. pavlovskyi,
I. tanuki, and I. turdus, were negative for rickettsiae infection. The highest infection
rate was observed in I. nipponensis (80.0%), followed by H. longicornis (62.8%),
I. monospinosus (58.6%), H. hystricis (57.8%), I. persulcatus (34.8%), A.
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testudinarium (23.5%), H. megaspinosa (17.4%), H. flava (10.2%), H. japonica
(5.1%), H. kitaokai (4.1%), H. formosensis (2.8%), and I. ovatus (1.1%).
3.2 gltA genotyping
Out of 373 samples that tested positive for rickettsiae by real-time PCR for
gltA, 352 samples yielded amplicons by conventional PCR for gltA, while 21
samples did not (Table I-2). All the amplicons were successfully sequenced, which
resulted in 15 different gltA genotypes (Figure I-2 and Table I-3). In the present
study, the gltA genotype is defined as a gltA sequence type that is different from the
others even by a single nucleotide. All gltA genotypes (G1, G2, G6, G7, G9, G11,
G12, G14, and G15) detected in the genus Haemaphysalis were clustered in the
same clade, while five genotypes (G3, G4, G5, G10, and G13) obtained from the
genus Ixodes were allocated to three different clusters while only one genotype (G8)
was linked with the genus Amblyomma (Figure I-3). A total of 13 genotypes were
detected in only one single tick species, while two genotypes (G5 and G11) were
detected in two different tick species: G5 was recovered from I. persulcatus and I.
monospinosus, and G11 was from H. japonica and H. flava (Figure I-3).
3.3 Geographic information on gltA genotypes and host ticks
Table I-3 represents the relationship between gltA genotypes and their
geographical origins. Out of 15 genotypes, 11 genotypes (G1, G2, G3, G4, G5, G6,
G8, G10, G11, G12, and G15) were detected from multiple geographical regions.
The other 4 genotypes (G7, G9, G13, and G14) were detected in only one single
tick species from a single region. G7 and G14 were detected in H. formosensis from
Kyusyu region, while G9 and G13 were in H. kitaokai from Kansai region and I.
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ovatus from Tohoku region, respectively (Figure I-1). The present study employed
H. formosensis and H. kitaokai from three different regions and I. ovatus collected
from five different regions (Table I-3).
3.4 Multiple genes sequencing
To further characterise Rickettsia spp. based on five other genes (ompA,
ompB, sca4, htrA, and 16S rRNA), PCR analyses were conducted on the selected
samples of each gltA genotype. A total of 57 samples were employed in this
analysis. We selected more than two samples from each genotype except for G7
which was found in only one sample (Table I-4). The samples with higher rickettsial
burden were selected based on the results of gltA real-time PCR. The mean
rickettsial burden in the template DNA ranged from 2.3E+2 to 2.1E+4 copies/μL
(Table I-4). The htrA gene was successfully amplified and sequenced for all gltA
genotypes (Table I-4). Although 16S rRNA PCR gave amplicons in all gltA
genotypes, the following sequencing analysis revealed that rickettsial 16S rRNA
gene sequences were obtained in only 12 gltA genotypes. The ompB, ompA and
sca4 genes were amplified and sequenced in 11, five and six different gltA
genotypes, respectively. All genes were successfully sequenced in two gltA
genotypes (G6 and G7). Four genes were successfully amplified in six gltA
genotypes (G1, G2, G5, G8, G10, and G11), and three genes were amplified in four
gltA genotypes (G3, G4, G9 and G13). Only the htrA gene was amplified in three
gltA genotypes (G12, G14, and G15) (Table I-4). The sequencing analysis of the
amplified products revealed that there were no sequence differences in any of the
genes in the samples with the same gltA genotypes. The sequence types obtained
from each gltA genotype were different from each other.
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3.5 Species classification of SFG rickettsiae
Phylogenetic trees inferred from ompA, ompB, sca4, htrA, and 16S rRNA
analysis are shown in Figure I-4, Figure I-5, Figure I-6, Figure I-7 and Figure I-8,
respectively. G4 and G5 formed a distinct cluster with R. helvetica in all trees when
sequences were available and thus were identified as R. helvetica. Being supported
by more than three trees, G13, G10, G8, and G3 were identified as R. asiatica, R.
monacensis (former Rickettsia sp. In56), and R. tamurae, and Candidatus R.
tarasevichiae, respectively (Table I-5). The other nine gltA genotypes could not be
classified into specific species due to a lack of consensus between the trees and/or
absence of sequences from previously validated rickettsial species in the same
phylogenetic cluster.
4 DISCUSSION
The present chapter included a total of 2,189 individual ticks collected at
114 different sampling sites in six regions of Japan for the screening of SFG
rickettsiae. Our nationwide sampling enabled us to collect as many as 19 different
tick species from four genera, most of which were common tick species prevalent
in Japan. A first screening test using gltA real-time PCR revealed that 17.0% (373
out of 2,189) of the ticks were infected with SFG rickettsiae. This infection rate was
comparative to the results of an earlier study where 21.9% (181 out of 827) of the
ticks, including 10 different species collected from central (Shizuoka, Mie, and
Wakayama prefectures) and southern (Kagoshima, Nagasaki, and Okinawa
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prefectures) parts of Japan, were positive for SFG rickettsiae (Gaowa et al., 2013).
Another nationwide survey conducted in 5 prefectures (Chiba, Hokkaido, Kochi,
Tokushima, and Toyama prefectures) including JSF-endemic areas reported an
overall positive rate for SFG rickettsiae to be 25.8% (186 out of 722) in 10 different
tick species (Ishikura et al., 2003).
Partial sequences of the gltA gene of SFG rickettsiae were determined by
conventional PCR, which was previously designed to characterise SFG rickettsiae
in Japan (Gaowa et al., 2013). Based on the sequences of the gltA gene obtained
from 352 ticks, the SFG rickettsiae detected in the present study were provisionally
divided into 15 genotypes (Figure I-2). In the molecular classification of SFG
rickettsiae, the analysis of multiple genes commonly used by other researchers is a
prerequisite (Fournier et al., 2003). Therefore, further analyses to obtain the
sequences of five additional genes, ompA, ompB, sca4, htrA, and 16S rRNA, were
conducted. These efforts lead to the identification of four validated rickettsial
species, namely R. asiatica, R. helvetica, R. monacensis, and R. tamurae, and the
provisional species Candidatus R. tarasevichiae (Figure I-4, Figure I-5, Figure I-6,
Figure I-7 and Figure I-8).
Prior to this study, there was no official report of the presence of R.
monacensis in Japan. A recent study indicated that Rickettsia sp. In56, a rickettsial
stain reported from ticks in Japan (Ishikura et al., 2003), might be a synonymous of
R. monacensis (Kim et al., 2013). Although several isolates of Rickettsia sp. In56
have been obtained from Japanese ticks (Fujita et al., 2013), lack of their sequence
information prevents a direct comparison between Rickettsia sp. In56 and R.
monacensis reported elsewhere. Nevertheless, the sequence analysis of multiple
genes (gltA, ompA, ompB, htrA, and 16S rRNA) conducted in the present study
18
confirmed the presence of R. monacensis in Japan (Figure I-2, Figure I-4, Figure I-
5, Figure I-7 and Figure I-8). R. monacensis was initially isolated from I. ricinus
collected from the English Garden in Germany using ISE6 cells (Simser et al.,
2002) and has been detected from the same tick species in Europe and neighbouring
countries (Sréter-Lancz et al., 2005; Milhano et al., 2010; Špitalská et al., 2014;
Venclikova et al., 2014; Biernat et al., 2016). I. nipponensis and I. sinensis are
considered as main vectors of R. monacensis in China and Korea, respectively (Ye
et al., 2014; Shin et al., 2013). In our study, R. monacensis was detected from four
I. nipponensis samples collected in the Tohoku and Kansai regions, while none of
the other tick species carried R. monacensis (Figure I-3 and Table I-3). These results
may suggest the relatively wide distribution of R. monacensis and a strong
association of R. monacensis with I. nipponensis in Japan. This SFG rickettsiae
caused Mediterranean spotted fever-like symptoms in humans in several countries
(Jado et al., 2007; Madeddu et al., 2012). More recently, the agent was isolated from
the blood of a patient with an acute febrile illness in Korea (Kim et al., 2017). Thus,
clinicians should be aware of R. monacensis as a possible cause of non-JSF
rickettsiosis in Japan.
Although SFG rickettsiae with each prospective gltA genotype were
analysed in further detail by sequencing five additional rickettsial genes, ompA,
ompB, sca4, htrA, and 16S rRNA, the amplification was not successful for some
genes (Table I-4). The ompA and sca4 genes were amplified only from one third of
the tested gltA genotypes. Considering the relatively high rickettsial abundance in
tested samples (Table I-4). PCR failure is either because some of the SFG rickettsiae
lack these genes as shown in TG rickettsiae that do not possess ompA gene
(Ngwamidiba et al., 2006), or because there are nucleotide mismatches in the primer
19
annealing sites. PCR failures of variable genes such as ompA, ompB, and sca4 are
common issues in the genetic characterisation of SFG rickettsiae (Ngwamidiba et
al., 2006; Nakao et al., 2013). Thus further attempts including the development of
universal primers and/or bacterial isolation followed by whole genome sequencing
are required to determine the phylogenetic positions of uncharacterised Rickettsia
spp.
In a previous nationwide survey of SFG rickettsiae conducted in Japan,
Gaowa et al. (2013) classified the detected rickettsiae (n = 181) into five groups
(Group 1–5) based on the gltA sequences (Gaowa et al., 2013). Groups 1 and 2 were
respectively identified as R. japonica and R. tamurae, whereas groups 3, 4, and 5,
showing high sequence similarity with Rickettsia sp. LON-13, R. raoultii, and
Candidatus R. principis, respectively, were not classified as validated rickettsial
species (Gaowa et al., 2013). In agreement with their report, gltA sequences
corresponding to groups 3 (G6), 4 (G2), and 5 (G1, G11, G12, G14, and G15) were
detected (Figure I-2). Unfortunately, limited information is available about these
uncharacterised Rickettsia spp. In our study, G6 and G2 were respectively detected
in H. longicornis and H. hystricis with high infection rates (62.8% and 57.8%,
respectively) (Table I-3), warranting further studies on the effect of these infections
for the survival and reproductive fitness of their hosts.
Two gltA genotypes (G7 and G9) were allocated into distinct clusters from
Rickettsia spp. previously reported from Japan (Figure I-2). G7 and G9 showed the
highest gltA sequence similarity with Rickettsia spp. reported from Kenya
(KT257873) and Hungary (EU853834), respectively. Rickettsia sp. reported from
Kenya was detected in Rhipicephalus maculatus (Mwamuye et al., 2017), while one
that from Hungary was detected in H. inermis and was provisionally named as
20
Candidatus R. hungarica (Hornok et al., 2010). Since the sequences of other genes
were not available from those Rickettsia spp., it was difficult to evaluate the degree
of genetic relatedness in more detail. Nonetheless, the presence of closely related
species in two geographically remote areas may indicate the worldwide distribution
of these poorly characterised SFG rickettsiae. Since the present study provided the
sequences of multiple genes of those rickettsiae, the information is useful in the
classification of SFG rickettsiae.
In the present study, a strong association between rickettsial genotypes and
their host tick species was detected, where 13 out of 15 gltA genotypes were
detected in only one single tick species (Figure I-3 and Table I-4). Furthermore,
there was minimal geographical restriction for the 11 gltA genotypes that were
recovered from multiple geographical regions (Table I-3). These observations may
indicate that most of the SFG rickettsiae species are found in ticks but not in
vertebrate hosts in the natural environment. However, further examinations are
needed to confirm this hypothesis by observing transstadial and transovarial
transmission of these SFG rickettsiae in ticks. The effect of these rickettsial
infections on tick physiology and reproduction remains to be elucidated.
Although the sampling was conducted at several JSF-endemic areas in Mie,
Kagoshima, and Kumamoto prefectures, none of the ticks were infected with R.
japonica. Considering the low level of genomic plasticity within R. japonica
isolates (Akter et al., 2017), it was hardly expected that a real-time PCR assay of
gltA would result in false-negatives. The positive rate of R. japonica infection in
the questing ticks was as low as 0.86% (18 out of 2,099), even in endemic areas as
is the case in Shimane prefecture (Tabara et al., 2011). Collectively, the failure in
the detection of R. japonica might be partly attributed to the sample selection
21
procedure with which only a maximum of 10 individual ticks per species, stage/sex,
and site were tested for SFG rickettsiae infection. Therefore, it should be noted that
the present study might not fully disclose the diversity of SFG rickettsiae in Japan,
which warrants further investigations by employing a larger number of samples.
22
5 SUMMARY
Spotted fever group (SFG) rickettsiae are obligate intracellular Gram-
negative bacteria mainly associated with ticks. In Japan, since the discovery of R.
japonica as the causative agent of Japanses spotted fever (JSF), five other SFG
rickettsiae, namely Rickettsia asiatica, Rickettsia heilongjiangensis, Rickettsia
helvetica, Rickettsia tamurae, and Candidatus R. tarsevichiae have been reported.
Additionally, previous studies have indicated the presence of other Rickettsia
species/genotypes in animals and questing ticks; however, their phylogenetic
position and pathogenic potential are poorly understood. To understand the overall
diversity of SFG rickettsiae and associated tick species in Japan, a nationwide cross-
sectional survey was conducted on ticks collected from 114 different sites in 12
prefectures. Out of 2,189 individuals (19 tick species in 4 genera), 373 (17.0%)
samples were positive for Rickettsia spp. as ascertained by real-time PCR
amplification of gltA gene. Conventional PCR and sequencing analyses of gltA
indicated the presence of 15 different genotypes of SFG rickettsiae. Further
characterisation based on the analysis of five additional genes, ompA, ompB, sca4,
htrA, and 16S rRNA, led to the identification of R. asiatica, R. helvetica, R.
monacensis (formerly reported as Rickettsia sp. In56 in Japan), R. tamurae, and
Candidatus R. tarasevichiae. Furthermore, several uncharacterised Rickettsia spp.
including ones showing high similarities with those designated as novel Rickettsia
spp. detected in geographically remote countries such as Kenya and Hungary were
discovered. A strong association between rickettsial genotypes and their host tick
species was observed, while there was little association between rickettsial
genotypes and their geographical origins. These observations may indicate that
23
most of the SFG rickettsiae have a limited host range and are maintained in certain
ticks in the natural environment. Further investigations on the potential roles of
these SFG rickettsiae on ticks are required to understand the mechanisms
underlying widespread existence of genetically variable rickettsiae in ticks. It is also
of importance to further evaluate pathogenic potential of these SFG rickettsiae to
humans and animals.
24
Table I-1. Primers uesd in the present study.
Primer Sequence (5'-3') Target gene Annealing temperature (°C) Amplicon size (bp) Reference
CS-F TCGCAAATGTTCACGGTACTTT citrate synthase gene (gltA) 60 74 Steno et al., 2005
CS-R TCGTGCATTTCTTTCCATTGTG
CS-P TGCAATAGCAAGAACCGTAGGCTGGATG
gltA_Fc CGAACTTACCGCTATTAGAATG citrate synthase gene (gltA) 55 580 Gaowa et al., 2013
gltA_Rc CTTTAAGAGCGATAGCTTCAAG
Rr.190.70p ATGGCGAATATTTCTCCAAAA outer membrane A gene (ompA) 48 542 Regnery et al., 1991
Rr.190.602n AGTGCAGCATTCGCTCCCCCT
120_2788 AAACAATAATCAAGGTACTGT outer membrane B gene (ompB) 48 816 Roux and Raoult, 2000
120_3599 TACTTCCGGTTACAGCAAAGT
D1f ATGAGTAAAGACGGTAACCT surface cell antigen-4 (sca4) 50 928 Sekeyova et al., 2001
D928r AAGCTATTGCGTCATCTCCG
sca4_seq1 GCCGGCTATTTCTATTGATTC* This study
sca4_seq2 TGCAAGCGATCTTAGAGCAA* This study
17k_5 GCTTTACAAAATTCTAAAAACCATATA 17-kDa common antigen gene (htrA) 52 550 Labruna et al., 2004
17k_3 TGTCTATCAATTCACAACTTGCC
Rick_16S_F3 ATCAGTACGGAATAACTTTTA 16S ribosomal RNA gene (16S rRNA) 52 1328 Anstead et al., 2013
Rick_16S_F4 TGCCTCTTGCGTTAGCTCAC
rrs_seq1 AGGCCTTCATCACTCACTCG*
This study
rrs_seq2 CTACACGCGTGCTACAATGG*
*The primers were used for sequencing.
25
Table I-2. Detection of spotted fever group rickettsiae by real-time and conventional PCR for gltA gene.
Tick species
No. tested
(Female/ Male/ Nymph)
Real-time PCR Conventional PCR
No. of positive
(Female/ Male/ Nymph) (%)
No. of positive
(Female/ Male/ Nymph) (%)
A. testudinarium 85 (3/0/82) 20 (1/0/19) (23.5) 16 (1/0/15) (18.8)
D. taiwainensis 12 (7/5/0) 0 0
H. concinna 7 (2/5/0) 0 0
H. cornigera 1 (1/0/0) 0 0
H. flava 128 (59/65/4) 13 (7/5/1) (10.2) 11 (6/4/1) (8.6)
H. formosensis 253 (130/122/1) 7 (2/5/0) (2.8) 7 (2/5/0) (2.8)
H. japonica 78 (50/25/3) 4 (2/2/0) (5.1) 4 (2/2/0) (5.1)
H. hystricis 64 (42/21/1) 37 (24/13/0) (57.8) 36 (23/13/0) (56.3)
H. kitaokai 74 (37/36/1) 3 (0/2/1) (4.1) 3 (1/1/1) (4.1)
H. longicornis 86 (56/26/4) 54 (31/22/1) (62.8) 54 (31/22/1) (62.8)
H. megaspinosa 201 (106/92/3) 35 (21/14/0) (17.4) 27 (16/11/0) (13.4)
H. yeni 1 (1/0/0) 0 0
I. monospinosus 58 (38/20/0) 34 (20/14/0) (58.6) 34 (20/14/0) (58.6)
I. nipponensis 5 (0/5/0) 4 (0/4/0) (80) 4 (0/4/0) (80)
I. ovatus 652 (339/313/0) 7 (4/3/0) (1.1) 7 (4/3/0) (1.1)
I. pavlovskyi 33 (16/17/0) 0 0
I. persulcatus 446 (220/222/4) 155 (87/68/0) (34.8) 150 (82/68/0) (33.6)
I. tanuki 2 (1/1/0) 0 0
I. turdus 3 (3/0/0) 0 0
Total 2,189 (1,111/975/103) 373 (199/152/22) (17.0) 352 (187/147/18) (16.1)
26
Table I-3. Host ticks and geographic origin of 15 gltA genotypes of spotted fever group rickettsiae.
gltA genotype Tick species No. of positive / No. tested (%)
Hokkaido Tohoku Chubu Kansai Kyushu Okinawa Total
G1 H. megaspinosa 5/94 (5.3) 0/2 (0) - 14/97 (14.4) 8/8 (100) - 27/201 (13.4)
G2 H. hystricis ˗ - - 5/8 (62.5) 31/53 (58.5) 0/3 (0) 36/64 (56.3)
G3 I. persulcatus 44/376 (11.7) 2/51 (3.9) 0/11 (0) 0/8 (0) - - 46/446 (10.3)
G4 I. persulcatus 96/376 (25.5) 0/51 (0) 1/11 (9.1) 0/8 (0) - - 97/446 (21.7)
G5 I. persulcatus 7/376 (18.6) 0/51 (0) 0/11 (0) 0/8 (0) - - 7/446 (1.6)
G5 I. monospinosus - 34/58 (58.6) - - - - 34/58 (58.6)
G6 H. longicornis 0/4 (0) 0/2 (0) 5/5 (100) 49/61 (80.3) 0/14 (0) - 54/86 (62.8)
G7 H. formosensis - - - 0/34 (0) 1/216 (0.5) 0/3 (0) 1/253 (0.4)
G8 A. testudinarium - - - 11/64 (17.2) 4/20 (20.0) 1/1 (100) 16/85 (18.8)
G9 H. kitaokai - - ˗ 2/43 (4.7) 0/14 (0) 0/17 (0) 2/74 (2.7)
G10 I. nipponensis ˗ 2/3 (66.7) ˗ 2/2 (100) ˗ ˗ 4/5 (80.0)
G11 H. japonica 2/49 (4.1) 2/27 (7.4) ˗ 0/2 (0) ˗ ˗ 4/78 (5.1)
G11 H. flava ˗ 3/28 (10.7) ˗ 4/71 (5.6) 1/29 (3.4) ˗ 8/128 (6.3)
G12 H. flava ˗ 1/28 (3.6) ˗ 2/71 (2.8) 0/29 (0) ˗ 3/128 (2.3)
G13 I. ovatus 0/463 (0) 7/163 (4.3) 0/10 (0) 0/15 (0) 0/1 (0) ˗ 7/652 (1.1)
G14 H. formosensis ˗ ˗ ˗ 0/34 (0) 2/216 (0.9) 0/3 (0) 2/253 (0.8)
G15 H. formosensis ˗ ˗ ˗ 1/34 (2.9) 3/216 (1.4) 0/3 (0) 4/253 (1.6)
-, This tick species was not collected in the region.
27
Table I-4. Results of PCR amplification for the ompA, ompB, htrA, sca4 and 16S rRNA genes.
gltA
genotype Tick species No. tested
Mean rickettsial
burden (copies/µl)*
PCR amplification
ompA ompB sca4 htrA 16S rRNA
G1 H. megaspinosa 2 7.9E+3 - + + + +
G2 H. hystricis 2 1.1E+4 - + + + +
G3 I. persulcatus 4 8.7E+3 + - - + +
G4 I. persulcatus 3 8.4E+3 - + - + +
G5 I. persulcatus 6 1.3E+3 - - - + -
G5 I. monospinosus 6 2.3E+2 - + + + +
G6 H. longicornis 2 2.4E+3 + + + + +
G7 H. formosensis 1 1.0E+4 + + + + +
G8 A. testudinarium 3 2.1E+4 + + - + +
G9 H. kitaokai 2 1.6E+4 - + - + +
G10 I. nipponensis 2 3.0E+3 + + - + +
G11 H. japonica 3 2.6E+3 - + + + +
G11 H. flava 7 1.8E+3 - - - + -
G12 H. flava 3 1.2E+3 - - - + -
G13 I. ovatus 5 2.5E+3 - + - + +
G14 H. formosensis 3 3.6E+3 - - - + -
G15 H. formosensis 3 1.3E+3 - - - + -
+, Amplified; -, Not amplified.
*The mean copy number of rickettsial gltA gene in the template DNA was calculated by gltA real-time PCR.
28
Table I-5. Classification of SFG rickettsiae and their related tick species.
gltA genotype Rickettsia species Related tick species
G1 Uncharacterised Rickettsia sp. H. megaspinosa
G2 Uncharacterised Rickettsia sp. H. hystricis
G3 Candidatus R. tarasevichiae I. persulcatus
G4 Rickettsia helvetica I. persulcatus
G5 Rickettsia helvetica I. persulcatus
G5 Rickettsia helvetica I. monospinosus
G6 Uncharacterised Rickettsia sp. H. longicornis
G7 Uncharacterised Rickettsia sp. H. formosensis
G8 Rickettsia tamurae A. testudinarium
G9 Uncharacterised Rickettsia sp. H. kitaokai
G10 Rickettsia monacensis I. nipponensis
G11 Uncharacterised Rickettsia sp. H. japonica
G11 Uncharacterised Rickettsia sp. H. flava
G12 Uncharacterised Rickettsia sp. H. flava
G13 Rickettsia asiatica I. ovatus
G14 Uncharacterised Rickettsia sp. H. formosensis
G15 Uncharacterised Rickettsia sp. H. formosensis
29
Figure I-1. A map of the 114 sample collection sites in this study.
30
Figure I-2. A phylogenetic tree of spotted fever group rickettsiae based on the
gltA gene sequences. The analysis was performed using a maximum likelihood
method with the Kimura two-parameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences detected in this
study are indicated in red. The number of samples positive for each genotype is
indicated in the parentheses.
31
Figure I-3. Relationship between spotted fever group rickettsiae and their related tick species. The left side of the tree shows a
phylogenetic tree of spotted fever group rickettsiae based on gltA gene sequence. The right side of phylogenetic tree represents a
simplified tick phylogeny consisting 12 tick species. The colour of the arrows indicates the link between rickettsial genotypes and
different tick species.
32
Figure I-4. Phylogenetic tree based on the sequences of the ompA gene of
spotted fever group rickettsiae. The analyses were performed using a maximum
likelihood method with the Kimura two-parameter model. All bootstrap values from
1,000 replications are shown on the interior branch nodes. The sequences obtained
in this study are shown in red.
33
Figure I-5. Phylogenetic tree based on the sequences of the ompB gene of
spotted fever group rickettsiae. The analyses were performed using a maximum
likelihood method with the Kimura two-parameter model. All bootstrap values from
1,000 replications are shown on the interior branch nodes. The sequences obtained
in this study are shown in red.
34
Figure I-6. Phylogenetic tree based on the sequences of the sca4 gene of spotted
fever group rickettsiae. The analyses were performed using a maximum likelihood
method with the Kimura two-parameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
35
Figure I-7. Phylogenetic tree based on the sequences of the htrA gene of spotted
fever group rickettsiae. The analyses were performed using a maximum likelihood
method with the Kimura two-parameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
36
Figure I-8. Phylogenetic tree based on the sequences of the 16S rRNA coding
gene of spotted fever group rickettsiae. The analyses were performed using a
maximum likelihood method with the Kimura two-parameter model. All bootstrap
values from 1,000 replications are shown on the interior branch nodes. The
sequences obtained in this study are shown in red.
37
CHAPTER II
Isolation of Rickettsia, Rickettsiella, and Spiroplasma from questing ticks in
Japan using arthropod cells
38
1 INTRODUCTION
Ticks are important vectors among blood sucking ectoparasites that
transmit various zoonotic pathogens to humans and animals through their bite.
Ticks harbour not only pathogenic microorganisms of veterinary and medical
importance (Jongejan and Uilenberg, 2004) but also several endosymbionts of the
genera Coxiella, Francisella, and Rickettsia (Paddock et al., 2004; Ahantarig et al.,
2013). The recent development of deep sequencing technologies has enabled high-
throughput screening of pathogens and symbionts in ticks and expanded our
knowledge on the diversity of microorganisms harboured by ticks (Nakao et al.,
2013; Qiu et al., 2014; Kurilshikov et al., 2015). Although these studies have led to
the discovery of several previously unexpected or poorly characterised
microorganisms, it is challenging to evaluate their roles in ticks and their pathogenic
potential to animals solely based on their partial genome sequences.
In Japan, several tick-borne human diseases have been recognised. Until
the recent emergence of severe fever with thrombocytopenia syndrome (SFTS)
(Takahashi et al., 2014) and the re-emergence of tick-borne encephalitis (TBE)
(Yoshii et al., 2017), most cases of tick-borne human diseases have been associated
with bacterial infections. In particular, Japanese spotted fever (JSF) caused by
Rickettsia japonica is the most common tick-borne human diseases with hundreds
of cases ported annually (National Institute of Infectious Diseases 2017). Several
other rickettsioses caused by Rickettsia heilongjiangensis, Rickettsia helvetica, and
Rickettsia tamurae have also been reported to date (Noji et al., 2005; Ando et al.,
2010; Imaoka et al., 2011). The etiological agents of these rickettsial diseases were
isolated from patients and ticks primarily using L929 mouse fibroblast cells (Uchida
39
et al., 1992; Fournier et al., 2002; Fujita et al., 2006; Mahara, 2006; Ando et al.,
2010; Andoh et al., 2014). However, none of these studies have used arthropod cells
for isolation of tick-borne pathogens.
At present, tick cell lines are indispensable tools to study the interaction
between ticks and tick-borne microorganisms including pathogens and symbionts
in vitro (Bell-Sakyi et al., 2007; Bell-Sakyi et al., 2012). The cells have also been
successfully used for isolating and propagating a number of tick-borne
microorganisms (Bell-Sakyi et al., 2007; Bell-Sakyi et al., 2015). For example, the
previously unculturable Borrelia lonestari was isolated and propagated for the first
time in a tick cell line (Varela et al., 2004). Similarly, other tick-borne pathogens
from genera such as Anaplasma and Ehrlichia have been successfully cultivated
and maintained in tick cell lines (Munderloh et al., 2003; Zweygarth et al., 2013).
The main goal of this chapter was to isolate and characterise tick-borne
microorganisms from field-collected ticks using two arthropod cell lines derived
from Ixodes scapularis embryo (ISE6) and Aedes albopictus larvae (C6/36). To
further characterize these isolates, four rickettisal genes (16SrRNA, gltA, opmA and
ompB) of rickettsiae were amplified and sequenced. As a result, five rickettsial
genotypes including four previously validated rickettsial species, one
uncharacterised rickettsial genotype and two tick endosymbionts, Rickettsiella and
Spiroplasma were isolated in an arthropod cell lines.
40
2 MATERIALS AND METHODS
2.1 Tick samples
This study employed unfed ticks collected at 11 different prefectures by a
flagging method between 2013 and 2015 (Table II-1). Tick species were identified
morphologically under a stereomicroscope using standard keys (Yamaguti et al.,
1971; Nakao et al.,1992). The samples included fifteen different tick species;
Amblyomma testudinarium (n = 10), Dermacentor taiwanensis (n = 3),
Haemaphysalis concinna (n = 3), H. flava (n = 8), H. formosensis (n = 15), H.
hystricis (n = 24), H. japonica (n = 9), H. kitaokai (n = 3), H. longicornis (n = 18),
H. megaspinosa (n = 27), Ixodes monospinosus (n = 7), I. nipponensis (n = 2), I.
ovatus (n = 9), I. pavlovskyi (n = 2) and I. persulcatus (n = 30). After identifying
tick species, ticks were washed with 70% ethanol and sterile PBS, then
homogenised in 100 µl of high-glucose Dulbecco’s Modified Eagle medium
(DMEM Gibco, Life Technologies, Carlsbad, CA, USA) by using Micro Smash
MS100 (TOMY, Tokyo, Japan). Half of the homogenate was subjected to DNA
extraction using a blackPREP Tick DNA/RNA Kit (Analytikjena, Germany), while
the other half was kept at -80°C and used for this study. A total of 170 tick
homogenates including 158 from single unfed tick (adult or nymph) and 12 from
nymphal pools (5 to 24 nymphs/pool) were included in the present study.
2.2 Maintenance of cell lines
ISE6 cells, originally reported by Kurtti et al. (1996) and received from the
CEH Institute of Virology and Environmental Microbiology (Oxford, UK), were
grown in L-15B medium supplemented with 10% foetal bovine serum, and 5%
41
tryptose phosphate broth (Sigma-Aldrich, St. Louis, MO, USA) at 32°C as
described previously (Munderloh and Kurtti, 1989), except that 0.1% bovine
lipoprotein concentrate was not included in the culture medium. C6/36 cells,
purchased from the American Type Culture Collection (No. CRL-1660), were
grown in Minimum Essential Medium (MEM, Gibco) supplemented with 10%
foetal bovine serum, 2% MEM Non-essential amino acids (Gibco), 1% Sodium
Pyruvate 100 mM (Gibco), and 1% L-glutamine (Gibco) at 28°C in a humidified
atmosphere of 5% CO2 in air.
2.3 Co-culture with tick homogenates
ISE6 and C6/36 cells were seeded in 24-well culture plates and incubated
overnight. On the following day, 5 μl of each tick homogenate was inoculated into
separate wells of both cell lines. Culture medium was changed every three days for
C6/36 cells and once a week for ISE6 cells. At 2 weeks post-inoculation (pi), 100
μl of culture suspension was passaged into new wells containing uninfected cells.
Second and third passages were conducted in the same way as first at 4 and 6 weeks
pi, respectively. At 8 weeks pi, the experiment was terminated. All the bacterial
isolates obtained in this study were preserved at -80°C for downstream analysis.
Cell morphology was observed daily under an inverted microscope to detect
cytopathic effects presumably caused by bacterial infections. When contamination
of fungi or environmental bacteria was observed, the contaminated wells were
sterilised with 10% hypochlorous acid for more than 10 min to prevent the spread
of contamination to the neighbouring wells.
42
2.4 PCR
When the cells showed sign of bacterial infection, DNA was extracted
from 100 μl of parent culture suspension and/or first subculture using a Wizard
Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the
manufacturer’s instructions. In addition, cell suspensions from all wells at 4 and 8
weeks pi (from first and third subcultures, respectively) were subjected to DNA
extraction to detect possible bacterial infection whether or not morphological
changes were seen in cells. PCR was conducted using the primers, fD1 and Rp2 to
amplify eubacterial 16S ribosomal DNA (rDNA) (Weisburg et al., 1991). In order
to characterise rickettsial isolates, three additional genes were amplified: citrate
synthase gene (gltA) (Gaowa et al., 2013), 190-kDa outer membrane gene (ompA)
(Regnery et al., 1991) and 120-kDa outer membrane protein gene (ompB) (Roux
and Raoult, 2000). All PCR reactions were conducted in a 25 µl-reaction mixture
containing 2.5 µl of 10 × KOD Plus Neo PCR Buffer, 0.5 µl of a high-fidelity KOD-
Plus-Neo DNA polymerase (Toyobo), 200 nM of each primer, and 1.0 µl of
template DNA. PCR conditions were as follows: 40 cycles of denaturation (94°C,
15 sec), annealing (55°C for gltA, ompA, and 16S rDNA and 48°C for ompB, 30
sec) and extension (68°C, 30 sec for gltA, ompA, and ompB and 90 sec for 16S
rDNA). For some samples, we conducted TA-cloning using the pGEM-T vector
(Promega, Madison, WI) as described previously (Nakao et al., 2013). The
experimental procedures were approved by the Hokkaido University Safety
Committee on Genetic Recombination Experiments (No. 2017-046). All the primer
information is available in Table II-2. All negative control containing sterile water
instead of template DNA was included in all PCR assays.
43
2.5 Real-time PCR
Real-time PCR to detect gltA gene of SFG and TG rickettsiae was
conducted using the primers and probe shown in Table II-2. Reactions mixtures
were prepared using THUNDERBIRD Probe qPCR Mix (Toyobo, Osaka, Japan)
and the reactions were carried out in a C1000 Thermal Cycler with a CFX96 Real-
Time PCR Detection System (BioRad Laboratories, Hercules, CA) as described in
chapter I.
2.6 Sequencing and data analysis
All amplified PCR products were purified using a NucleoSpin Gel and
PCR Clean Up Kit (Takara Bio Inc.) and sequenced using the BigDye Terminator
version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). To
sequence 16S rDNA PCR products, sequencing primers were newly designed in the
present study (Table II-2). The purified sequencing products were analysed on an
ABI Prism 3130xl Genetic Analyzer Kit (Applied Biosystems) according to the
manufacturers' instructions. The sequences that were obtained were submitted to
the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp) under accession numbers
(16S rRNA: LC388759-LC388776; gltA: LC388777-LC388788; ompA:
LC388789-LC388795; and ompB: LC388796-LC388807). Sequenced data were
aligned using ATGC software version 6.0.4. Phylogenetic analyses were conducted
by a maximum likelihood method using MEGA7 version 7.0.18 (Kumar et al.,
2016). Bootstrap values were obtained with 1,000 replicates.
44
3 RESULTS
3.1 Real-time PCR
Among 170 tick homogenates, 114 were tested positive by real-time PCR
(Table II-1). Some of the samples were tested by real-time PCR in chapter I. For
the bacterial isolation, both rickettsiae-positive and -negative samples were used.
3.2 Isolation results
During the 8 weeks observation period, we confirmed bacterial isolation
in 14 and 4 different samples using ISE6 and C6/36 cells, respectively (Table II-3).
Ten isolates (9 from ISE6 and 1 from C6/36) were obtained in the parent cultures,
while 8 isolates (5 from ISE6 and 3 from C6/36) were obtained in the first
subcultures (Figure II-1). The sequencing analysis of the 16S rDNA PCR products
indicated that they were previously known tick-borne bacteria in three different
genera; Rickettsia, Rickettsiella, and Spiroplasma (Table II-4). Although a further
11 and 4 samples in ISE6 and C6/36 cells, respectively, also showed bacterial
growth in the well, sequencing analysis indicated the growth of environmental
bacteria such as Bacillus spp. Pseudomonas spp., and Mycobacterium spp. (data not
shown). Fungal infections developed in 75 ISE6 and 57 C6/36 wells; the remaining
77 and 98 wells were respectively did not yield any isolates (Table II-3). Although
4 rickettsial and 3 spiroplasmal isolates did not show any cytopathic effects in the
infected cells, their infections were detected by PCR conducted at 28 days pi (Figure
II-1).
45
3.3 Rickettsia isolation
Twelve isolates of Rickettsia (11 from ISE6 and 1 from C6/36) were
obtained from different tick homogenates (Table II-3). The amplification of gltA
and ompB genes was successful in all rickettsial isolates, while the ompA gene was
amplified only from seven isolates (Table II-4). Based on the phylogenetic analysis
of each rickettsial gene, the 12 isolates were identified as 4 previously validated
species, R. asiatica (n = 2), R. helvetica (n = 3), R. monacensis (n = 2), R. tamurae
(n = 3) and Rickettsia sp. LON, one uncharacterised rickettsial genotype, previously
isolated from H. longicornis in Japan (Fujita, 2008) (n = 2) (Figure II-2, Figure II-
3, Figure II-3, Figure II-4 and Figure II-5). There was a complete correspondence
between rickettsial species/genotype and tick species of origin; R. asiatica, R.
helvetica, R. monacensis, R. tamurae, and Rickettsia sp. LON were isolated from I.
ovatus, I. persulcatus, I. nipponensis, A. testudinarium, and H. longicornis,
respectively. A cytopathic effect was observed in R. helvetica-, R. monacensis-, and
R. tamurae-infected cells at 6, 14 and 10 days pi, while there was no obvious
morphological damage observed in R. asiatica- and Rickettsia sp. LON-infected
cells (Figure II-1).
3.4 Rickettsiella isolation
Rickettsiella was isolated from a homogenate of H. concinna collected in
Hokkaido using both ISE6 and C6/36 cells (Table II-1). A cytopathic effect was
observed at 6 and 13 days pi in ISE6 and C6/36 cells, respectively (Figure II-1).
The sequences of 16S rDNA of Rickettsiella obtained from two cell lines (Hcn-412I
and Hcn-412C) were identical and showed 100% identity with Rickettsiella sp.
detected from Ixodes uriae from Grimsey Island in Iceland (GenBank No.
46
KT697673). A phylogenetic analysis showed that our isolates formed a cluster with
Rickettsiella spp. detected from pea aphids (Figure II-6).
3.5 Spiroplasma isolation
Four Spiroplasma isolates were obtained (2 each from ISE6 and C6/36)
(Table II-3). Since two isolates were obtained from the same tick homogenate using
different cell lines, four isolates originated from three tick homogenates: I.
monospinosus, I. persulcatus, and H. kitaokai collected from Yamagata, Hokkaido
and Fukushima prefectures, respectively (Table II-1). Only one isolate (Ipe-147I)
showed a cytopathic effect in ISE6 cells at 23 days pi (Figure II-1). Among 4
isolates, two sequence types of 16S rDNA had two base pair (bp) differences in
their sequences. One sequence type was obtained from two isolates (Imo-135I and
Imo-135C) from I. monospinosus and one isolate (Hki-1033C) from H. kitaokai,
while the other was from I. persulcatus (Ipe-147I). Both sequences showed the
highest identities (1443/1444 bp and 1441/1444 bp) with 16S rDNA from
Spiroplasma sp. detected from Fannia manicata (little housefly) (GenBank No.
AY569829). In a phylogenetic analysis, these isolates were clustered together with
16S rDNA of two tick-derived Spiroplasma isolates; Spiroplasma ixodetis
(GenBank No. NR104852) obtained from Ixodes pacificus and Spiroplasma sp.
Bratislava 1 (GenBank No. KP967685) obtained from I. ricinus, and several
Spiroplasma spp. detected from various arthropods (Figure II-7).
47
4 DISCUSSION
Isolation and propagation of R. monacensis was firstly achieved using
ISE6 cells. This rickettsial agent has not been officially reported in Japan; in a
recent nationwide survey where R. monacensis infection was found in I.
nipponensis with high infection rates (2 positive out of 3 in the Tohoku region and
2 positive out of 2 in the Kansai region) (Chapter I). R. monacensis was first isolated
from Ixodes ricinus collected in Germany using ISE6 cells (Simser et al., 2002),
where the authors detected a cytopathic effect after the third passage (5 months). In
this experiment, a cytopathic effect was observed as early as 14 days after the
inoculation in both ISE6 and C6/36 cells (Figure II-1). Although the reason for this
difference is not clear, it may suggest that phenotypes under in vitro culture
conditions are different between R. monacensis strains. R. monacensis has been
associated with a human rickettsiosis presenting Mediterranean spotted fever-like
symptoms (Jado et al., 2007; Madeddu et al., 2012; Kim et al., 2017). The present
study reconfirmed the presence of this rickettsial agent in Japan and highlighted the
necessity for further investigation of the clinical cases it may cause.
In addition to R. monacensis, three previously validated rickettsial species,
R. asiatica, R. helvetica, R. tamurae, and one uncharacterised genotype, Rickettsia
sp. LON were isolated using ISE6 cells, among which only R. asiatica was also
isolated from C6/36 cells (Table II-4). Although the C6/36 cells inoculated with the
tick homogenates from which Rickettsia were isolated using ISE6 cells were tested
for rickettsial infections by PCR at 8 weeks pi, there was no positive amplicons.
However, when the isolates of R. monacensis and R. helvetica obtained from ISE6
culture were inoculated into C6/36 cells in preliminary experiments, their persistent
48
growth in the cells was observed (data not shown). In fact, R. monacensis and R.
helvetica were isolated from I. ricinus in Portugal using C6/36 cells in a previous
study (Milhano et al., 2010). These results may indicate that lower success rates of
rickettial isolation using C6/36 cells is partly attributed to low bacterial burden in
the inocula. It is also possible that propagation in ISE6 cells might help the
rickettsial isolates adapt to in vitro culture conditions using C6/36.
There are only a few reports on the in vitro propagation of Rickettsiella.
For example, Rickettsiella grylli was isolated from the variegated grasshopper,
Zonocerus variegatus, was cultured in several cell lines derived from different
arthropods (Henry et al., 1986). To the best of our knowledge, this is the first report
of the successful isolation of Rickettsiella spp. from ticks using ISE6 cells and
C6/36 cells. Bacteria within the genus Rickettsiella are known to be symbionts of
many arthropods (Tsuchida et al., 2010; Leclerque et al., 2011; Iasur-Kruh et al.,
2013; Łukasik et al., 2013). In ticks, Rickettsiella species have been reported in
genera Ixodes and Ornithodoros (Kurtti et al., 2002; Vilcins et al., 2009; Duron et
al., 2015; Duron et al., 2016). A recent metagenomic approach based on 16S rDNA
amplicons also showed the presence of Rickettsiella in the genus Haemaphysalis
(Khoo et al., 2016). The sequence analysis of 16S rDNA revealed that the
Rickettsiella sp. isolated from H. concinna showed 100% identity with Rickettsiella
endosymbiont detected in I. uriae collected from a seabird in Iceland (Figure II-6).
These facts may support the hypothesis that Rickettsiella has a wide geographical
distribution in ticks and is maintained by horizontal transfer between arthropod
species as previously suggested (Duron et al., 2016). However, a more detailed
analysis such as whole genome comparison between Rickettsiella spp. found in
different arthropod hosts is essential to prove this hypothesis. The role of
49
Rickettsiella in ticks is totally unknown; however, the lines of evidence from other
arthropods indicated an effect on the survival of their host arthropods (Tsuchida et
al., 2010; Łukasik et al., 2013). The isolate obtained in this chapter might be useful
to further investigate potential roles of Rickettsiella in ticks.
Four spiroplasmal isolates were obtained from three tick species: I.
monospinosus, I. persulcatus, and H. kitaokai. The isolates obtained from I.
monospinosus and H. kitaokai had completely identical 16S rDNA sequence and
all spiroplamal isolates in this chapter made one clade with previously reported
Spiroplasma species which were detected from a variety of arthropod including
ticks, ladybirds, plant hoppers, and mealybugs (Figure II-7). These findings may
suggest that Spiroplasma is maintained by horizontal transfer between different
arthropod species as suggested for Rickettsiella. This hypothesis should be explored
in future studies. Most members of Spiroplasma are symbionts in arthropods and
some of them are known to be beneficial to their hosts for example, by protecting
from fungal or parasitic infections (Łukasik et al., 2013; Xie et al., 2014; Yadav et
al., 2018). In some arthropods, Spiroplasma species are pathogenic and cause
gender-ratio distortions known as a male-killing effect in Drosophila (Harumoto et
al., 2014). However, a recent study conducted on a nidicolous tick Ixodes
arboricula did not find any association between female-biased sex ratios and
infections of six maternally inherited bacteria including Spiroplasma (Van Oosten
et al., 2018). Moreover, Spiroplasma mirum, an isolate obtained from rabbit tick
Haemaphysalis leporispalustris in the USA (Tully et al., 1982), was shown to have
potentially pathogenic properties; for example, S. mirum was virulent for chick
embryos and induced cataracts or lethal brain infections when introduced
intracerebrally into experimental animals such as suckling rats, rabbits (Tully et al.,
50
1977). Collectively, further biological characterisation of Spiroplasma detected in
the present study is necessary to understand their roles in ticks and potential risks
for human and animal health.
In chapter II, a number of wells were contaminated with bacterial or fungal
infections (79 wells of ISE6 culture and 78 wells of C6/36 culture) (Table II-3),
despite the fact that tick surface was washed with 70% ethanol and sterile PBS. This
result highlights the necessity of using additional chemicals to sterilize tick surface,
especially the ones effective for fungal infections. Another possible option might
be to use only internal organs for bacterial isolation by dissecting ticks.
51
5 SUMMARY
Ticks are blood sucking ectoparasites that transmit zoonotic pathogens to
humans and animals. Ticks harbour not only pathogenic microorganisms, but also
endosymbionts. Although some tick endosymbionts are known to be essential for
the survival of ticks, their roles in ticks remain poorly understood. The main aim of
this chapter was to isolate and characterise tick-borne microorganisms from field-
collected ticks using two arthropod cell lines derived from Ixodes scapularis
embryo (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick
homogenates originating from 15 different tick species collected in Japan were
inoculated into each cell line. Bacterial growth was confirmed by PCR
amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8 weeks
observation period, bacterial isolation was confirmed in 14 and 4 different samples
using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA
PCR products indicated that they were previously known tick-borne
pathogens/endosymbionts in three different genera; Rickettsia, Rickettsiella, and
Spiroplasma. These included 4 previously validated rickettsial species namely
Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n =
2), and Rickettsia tamurae (n = 3) and one uncharacterised genotype Rickettsia sp.
LON (n = 2). Four isolates of Spiroplasma had the highest similarity with
previously reported Spiroplasma isolates; Spiroplasma ixodetis obtained from ticks
in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in
Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella
sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our
knowledge, this is the first report on successful isolation of Rickettsiella from ticks.
52
The isolates obtained in this study can be further analysed to evaluate their
pathogenic potential to animals and their roles as symbionts in ticks.
53
Table II-1. Collection detail of ticks used for bacterial isolation using arthropod cells.
, This tick species was not collected in the study.
aNymphal pool samples were prepared from a pool of 5 to 24 nymphs.
bThe number in parentheses indicates the number of samples positive for rickettsiae by
gtlA realtime PCR.
Prefecture Collected year Tick species Female Male Nymph Nymphal
poola
Hokkaido 2013 H. concinna - 3 (0)b - -
2013 H. japonica 5 (0) 1 (1) - -
2013 & 2015 H. megaspinosa 3 (1) 10 (4) - 5 (5)
2013 I. ovatus 1 (0) 5 (1) - -
2015 I. pavlovskyi - 2 (0) - -
2013, 2014 &
2015 I. persulcatus 20 (18) 8 (5) - -
Fukushima 2014 D. taiwanensis - 1 (0) - -
2013 H. flava - 3 (2) - 1 (1)
2014 H. japonica 1 (1) 1 (1) - 1 (1)
2014 H. kitaokai 2 (0) 1 (0) - -
2014 H. megaspinosa - - - 1 (1)
2014 I. monospinosus - 1 (1) - -
2014 I. nipponensis - 1 (1) - -
2014 I. persulcatus 1 (1) 1 (0) - -
Yamagata 2014 H. flava 1 (1) - - 1 (1)
2013 & 2014 I. monospinosus 5 (5) 1 (1) - -
2013 I. nipponensis - 1 (1) - -
2014 I. ovatus 1 (1) 1 (1) - -
Tochigi 2014 H. flava - - - 1 (1)
Shizuoka 2013 H. longicornis 5 (5) - - -
Nara 2014 H. longicornis - - 1 (1)
2014 H. megaspinosa - 1 1
Wakayama 2015 A. testudinarium 1 (1) - - -
2015 D. taiwanensis 1 (0) 1 (0) - -
2015 H. formosensis 1 (0) - - -
2013 H. hystricis 4 (3) - - -
2013 & 2015 H. longicornis 3 (2) 8 (8) 1 (1) -
2015 H. megaspinosa 1 (1) - - -
2015 I. ovatus 1 (0) - - -
Kumamoto 2015 H. formosensis 4 (1) 3 (2) - -
2015 H. hystricis 1 (1) 2 (1) - -
Miyazaki 2013 A. testudinarium - - 6 (3) -
2013 H. flava 1 (1) - - -
2013 H. formosensis 1 (1) 2 (0) - -
2013 H. megaspinosa 2 (2) 1 (1) - -
Kagoshima 2015 A. testudinarium - - 2 (1) -
2015 H. formosensis - 4 (1) - -
2015 H. hystricis 5 (4) 12 (11) - -
2013 & 2015 H. megaspinosa 1 (1) 1 (1) - -
Okinawa 2014 A. testudinarium - - 1 (1) -
Total 72 (51) 76 (45) 10 (6) 12 (12)
54
Table II-2. Oligonucleotide primer pairs and probe used in real-time and conventional polymerase chain reactions and
sequencing.
Primer Primer sequence (5'-3') Target gene Target organism(s) Annealing temperature (°C)
Amplicon size (bp)
Reference
CS-F TCGCAAATGTTCACGGTACTTT citrate synthase gene (gltA) Rickettsiae spotted fever
group and typhus group 60 74 Stenos et al., 2005
CS-R TCGTGCATTTCTTTCCATTGTG
CS-P TGCAATAGCAAGAACCGTAGGCTGGATG
gltA_Fc CGAACTTACCGCTATTAGAATG citrate synthase gene (gltA) Rickettsia spp. 55 580 Gaowa et al., 2013
gltA_Rc CTTTAAGAGCGATAGCTTCAAG
Rr.190.70p ATGGCGAATATTTCTCCAAAA outer membrane A gene (ompA) Rickettsia spp. 55 632 Roux et al., 1996
Rr.190.701n GTTCCGTTAATGGCAGCATCT
120-2788 AAACAATAATCAAGGTACTGT outer membrane B gene (ompB) Rickettsia spp. 48 816 Roux and Raoult,
2000
120-3599 TACTTCCGGTTACAGCAAAGT
fD1 AGAGTTTGATCCTGGCTCAG 16S ribosomal RNA gene (16S
rRNA) Eubacteria 55 about 1500
Weisburg et al.,
1991
Rp2 ACGGCTACCTTGTTACGACTT
rrs_seq1 AGGCCTTCATCACTCACTCG* 16S rRNA of Rickettsia Rickettsia spp. This study
rrs_seq2 CTACACGCGTGCTACAATGG* 16S rRNA of Rickettsia, Spiroplasma and Rickettsiella
Rickettsia spp.,
Spiroplasma spp. and
Rickettsiella spp. This study
rrs_seq3 CGTGTCTCAGTCCCAATGTG* 16S rRNA of Spiroplasma Spiroplasma spp. This study
*The primers were used for sequencing.
55
Table II-3. Summary of bacterial isolation from homogenates of ticks
collected in Japan using tick (ISE6) and mosquito (C6/36) cell lines.
Isolation result Cell line
ISE6 C6/36
Rickettsia and symbionts 14 4
Bacterial contamination 4 11
Fungal contamination 75 57
No isolate 77 98
Total 170 170
56
Table II-4. Details of ticks from which bacterial isolates were obtained and the results of real-time and conventional PCRs amplifying
bacterial isolates.
ID Tick species Stage
/Sex Prefecture
Arthropod cell line Real-time
PCR resulta
PCR resultb
ISE6 C6/36 16S rRNA Rickettsial
gltA
Rickettsial
ompA
Rickettsial
ompB
135 I. monospinosus M Yamagata Isolated (Spiroplasma) Isolated (Spiroplasma) + + NA NA NA
141 I. nipponensis M Yamagata Isolated (Rickettsia) No isolate + + + + +
147 I. persulcatus F Hokkaido Isolated (Spiroplasma) Contaminated (Mycobacterium) + + NA NA NA
202 I. persulcatus M Hokkaido Isolated (Rickettsia) No isolate + + + - +
309 I. persulcatus M Hokkaido Isolated (Rickettsia) No isolate + + + - +
318 I. persulcatus M Hokkaido Isolated (Rickettsia) No isolate + + + - +
412 H. concinna M Hokkaido Isolated (Rickettsiella) Isolated (Rickettsiella) - + NA NA NA
772 A. testudinarium N Miyazaki Isolated (Rickettsia) Contaminated (7 dpi) + + + + +
774 A. testudinarium N Miyazaki Isolated (Rickettsia) No isolate + + + + +
1033 H. kitaokai F Fukushima No isolate Isolated (Spiroplasma) - + NA NA NA
1187 I. nipponensis M Fukushima Isolated (Rickettsia) No isolate + + + + +
1284 I. ovatus F Yamagata Isolated (Rickettsia) Contaminated (Bacillus) + + + - +
1328 I. ovatus M Yamagata Contaminated (2dpi) Isolated (Rickettsia) + + + - +
1994 A. testudinarium F Wakayama Isolated (Rickettsia) Contaminated (1 dpi) + + + + +
2014 H. longicornis M Wakayama Isolated (Rickettsia) No isolate + + + + +
2019 H. longicornis M Wakayama Isolated (Rickettsia) Contaminated (Williamsia) + + + + +
aScreened by genus specific realtime PCR using DNA extracted from the whole ticks. +, positive; , negative.
bPCR using DNA extracted from ISE6 and C6/36 cells. NA, not applicable; dpi, day post inoculation.
57
Figure II-1. The time of onset of cytopathic effect observed in each bacterial
isolate in ISE6 and C6/36 cells.
58
Figure II-2. Phylogenetic tree based on the sequences of 16S rRNA coding gene
of Rickettsia isolates. The analyses were performed using maximum likelihood
method with the Kimura twoparameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
59
Figure II-3. Phylogenetic tree based on of the sequences of the gltA gene of
Rickettsia isolates. The analyses were performed using maximum likelihood
method with the Kimura twoparameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
60
Figure II-4. Phylogenetic tree based on the sequences of ompA gene of
Rickettsia species. The analyses were performed using maximum likelihood
method with the Kimura twoparameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
61
Figure II-5. Phylogenetic tree based on the sequences of ompB gene of
Rickettsia isolates. The analyses were performed using maximum likelihood
method with the Kimura twoparameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
62
Figure II-6. Phylogenetic tree based on the sequences of 16S rRNA coding gene
of Rickettsiella isolates. The analyses were performed using maximum likelihood
method with the Kimura twoparameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
63
Figure II-7. Phylogenetic tree based on the sequences of 16S rRNA coding gene
of Spiroplasma isolates. The analyses were performed using maximum likelihood
method with the Kimura twoparameter model. All bootstrap values from 1,000
replications are shown on the interior branch nodes. The sequences obtained in this
study are shown in red.
64
GENERAL CONCLUSION
Rickettsiae are obligate intracellular Gram-negative bacteria that cause
rickettsioses in humans throughout the world. Ticks harbor most of the members of
spotted fever group (SFG) rickettsiae and transmit them to humans and animals. In
Japan, Rickettsia japonica, a causative agent of Japanese spotted fever (JSF), was
firstly identified as a human pathogen. Subsequently, several other rickettsioses
caused by Rickettsia heilongjiangensis, Rickettsia helvetica and Rickettsia tamurae
have also been reported; however, there is scanty information on the overall
diversity of Rickettsia species circulating in Japan. This thesis aimed to characterise
a wide range of SFG rickettsiae in vector ticks in Japan by analysing multiple
rickettsial genes which enables the detailed phylogenetic classification of SFG
rickettsiae and to isolate Rickettsia using arthropod cell lines.
In chapter I, 19 tick species collected from 114 different sites in 12
prefectures were tested for rickettsial infections. Out of 2,189 individuals, 373
(17.0%) samples were positive for Rickettsia spp. as ascertained by real-time PCR
amplification of gltA gene. Fifteen genotypes of SFG rickettsiae based on the
sequences of the gltA gene were detected from three tick genera; Amblyomma,
Ixodes and Haemaphysalis collected from different regions. There was a strong
association between rickettsial genotypes and their host tick species, indicating that
most of the SFG rickettsiae are maintained in certain tick species in the natural
environment. From the analysis of multiple rickettsial genes, five validated
rickettsial species, namely R. asiatica, R. helvetica, R. monacensis (formerly
reported as Rickettsia sp. In56 in Japan), R. tamurae, and Candidatus R.
tarasevichiae, were identified; however, only the limited number of ompA, ompB
65
and sca4 sequences were obtained from some of the SFG rickettsiae. There is need
for further studies of whole genome sequencing to investigate the poorly
characterised Rickettsia.
In chapter II, arthropod cell lines (C6/36 and ISE6) were employed to
isolate tick-borne pathogens including rickettsiae. A total of 170 tick homogenates
originating from 15 different tick species collected in Japan were employed. The
use of arthropod cell lines led to successful isolation of bacteria in three different
genera; Rickettsia, Rickettsiella, and Spiroplasma. These included 4 previously
validated rickettsial species such as R. asiatica, R. helvetica, R. monacensis, and R.
tamurae and one uncharacterised genotype Rickettsia sp. LON. Although the
technique needs to be improved to reduce contaminations by fungal infections, the
use of arthropod cell lines seems promising to expand the knowledge on
microorganisms in ticks.
The studies included in this thesis highlight the wide distribution and high
frequency of SFG rickettsiae in ixodid ticks and provide basic information essential
to understand epidemiology of rickettsiosis in Japan. The genetic information is
useful for future development of diagnostic methods specific for pathogenic
rickettsiae. The bacterial isolates are important to further analyse their pathogenic
potential in vertebrate animals and their roles as symbionts in ticks.
66
ACKNOWLEDGEMENTS
I would like to take this opportunity to acknowledge my enormous debt to
Professor Dr. Ken KATAKURA (Laboratory of Parasitology, Graduate School of
Infectious Diseases, Faculty of Veterinary Medicine, Hokkaido University) for his
kind suggestion to conduct the doctoral course in Hokkaido University. I am deeply
indebted to Associate Professor Dr. Ryo NAKAO (Laboratory of Parasitology,
Graduate School of Infectious Diseases, Faculty of Veterinary Medicine, Hokkaido
University) for his valuable supervising and research training, creating research
knowledge and concepts and correcting the manuscripts throughout the period of
my study.
I wish to convey special gratitude to my thesis advisors; Professor Dr.
Chihiro SUGIMOTO (Global Station for Zoonosis Control, Global Institution for
Collaborative Research and Education, Hokkaido University), Professor Dr.
Hiroaki KARIWA (Laboratory of Public Health, Department of Environmental
Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido
University), and Associate Professor Dr. Norikazu ISODA (Unit of Risk Analysis
and Management, Research Center for Zoonosis Control, Hokkaido University) for
their creditable supervision and valuable advice.
My special thanks are going to my senior Dr. Yongjin QIU (Hokudai Center
for Zoonosis Control in Zambia, School of Veterinary Medicine, University of
Zambia) for his admirable counseling, providing moral support whenever I needed
help and also for his lovingly care during the oversea practice in Zambia.
I would like to extend my appreciation to Dr. Oleg Mediannikov (Research
Unit of Tropical and Emerging Infectious Diseases, Institute of the Research for the
67
Development, Faculty of Medicine, Aix-Marseille University) for his special
lecture and inspiration for Rickettsia research during his visiting to Japan. My
special appreciation goes to Dr. Lesley Bell-Sakyi (The Tick Cell Biobank,
Department of Infection Biology, Institute of Infection and Global Health,
University of Liverpool) for her kindly acceptance of tick cells training and fruitful
explanation and scientific discussion about tick cell lines.
Special thanks are going to my respectful previous supervisors; Professor
Dr. Tin Tin Myaing, Professor Dr. Tin Ngwe, Professor Dr. Mar Mar Win,
Professor Dr. Lat Lat Htun and Associate Professor Dr. Saw Bawm (University of
Veterinary Science, Naypyidaw, Myanmar) for their kind recommendation and
suggestion to continue the study in Japan.
I would like to extend my thanks to Ms. Mayumi SASADA, Ms. Aiko
OHNUMA and Ms. Chiho KANEKO (Department of Veterinary Medicine, Faculty
of Agriculture, University of Miyazaki) for their technical supports at the beginning
of study and their kind encouragement during my stay in Sapporo. I wish to express
my special thanks to all collaborators who supported in collection of ticks in each
prefecture.
Many thanks are due to formal and present students and staffs of Laboratory
of Parasitology and Research Center for Zoonosis Control, Graduate School of
Veterinary Medicine for creating a pleasant and enjoyable social support.
I wish to express my sincere thanks to my beloved family members for their
understanding, endless love and moral support during this study. I will always be
grateful for their support.
Finally, I wish to thank the Japanese scholarship from the Ministry of
Education, Culture, Sports, Science and Technology MEXT for the finance of
68
living cost in Sapporo and the program of Fostering Global Leaders in Veterinary
Science toward Contributing to 'One Health' for the funding of oversea experience
during doctoral course.
69
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JAPANESE ABSTRACT
リケッチアはグラム陰性の細胞内寄生細菌で、しばしばヒトにお
いてリケッチア症の原因となる。多くの紅斑熱群リケッチアはマダニによ
ってヒトや動物に伝播される。本邦においては、Rickettsia japonica が
日 本 紅 斑 熱 の 病 原 菌 と し て 報 告 さ れ て 以 来 、 Rickettsia
heilongjiangensis、 Rickettsia helvetica、Rickettsia tamurae がヒ
トに病原性をもつ紅斑熱群リケッチアとして検出されてきた。一方で、ヒ
トへの病原性が不明な複数のリケッチア種および遺伝子型の存在が示唆さ
れており、本邦における紅斑熱群リケッチア種の全容を解明する必要があ
る。そこで、本研究では国内のマダニが保有する紅斑熱群リケッチアにつ
いて複数の遺伝子座の塩基配列解析により遺伝的多様性を評価すること、
ならびに節足動物由来細胞を用いて紅斑熱群リケッチアを含むマダニ保有
微生物を分離することを目的とした。
第一章では、国内の 114 地点で採集した 19種のマダニを対象に紅
斑熱群リケッチアの遺伝子検出を試みた。リアルタイム PCR による解析の
結果、2,189 個体のマダニのうち 373 個体(17.0%)で陽性反応を得た。ク
エン酸合成酵素遺伝子(gltA)の塩基配列に基づいた型別の結果、検出さ
れた紅斑熱群リケッチアは 15 の遺伝子型に分別された。リケッチア gltA
遺伝子型とマダニ種との間には強い関連性がみられたことから、ほとんど
の紅斑熱群リケッチアはそれぞれ特定のマダニ種によって自然界で維持さ
れていることが示唆された。さらに、複数のリケッチア遺伝子座の塩基配
85
列解析により、 Rickettsia asiatica, R. helvetica, Rickettsia
monacensis (Rickettsia sp. In56), R. tamurae, and Candidatus
Rickettsia tarasevichiae を特定した。一方で、いくつかのリケッチア
gltA遺伝子型では、外膜蛋白質 A 遺伝子(ompA)など複数の遺伝子で PCR
増幅がみられなかった。今後、全ゲノム解析などにより、これらの紅斑熱
群リケッチアの系統学的な位置関係を明らかにする必要がある。
第二章では、節足動物由来細胞(C6/36 および ISE6)を用いて、
紅斑熱群リケッチアを含むマダニ保有微生物の分離培養を試みた。15 種
のマダニに由来する 170 検体のマダニ乳剤を用いて、節足動物由来細胞と
の共培養を行った。その結果、16 週間の実験期間において、リケッチア
属、リケッチエラ属、スピロプラズマ属の細菌、合計 18 菌株の分離に成
功した。得られた分離株の遺伝子解析の結果、4 種のリケッチア属細菌
(R. asiatica、R. helvetica, R. monacensis、R. tamurae)と 1種のリ
ケッチア遺伝子型(Rickettsia sp. LON)が確認された。節足動物由来細
胞は紅斑熱群リケッチアを含む多様なマダニ保有微生物の分離培養に有用
であることが示された。
以上の結果から、本邦のマダニには遺伝的に多様な紅斑熱群リケ
ッチアが広汎に分布していることが明らかとなった。得られた成果は、本
邦におけるリケッチア症の疫学を理解する上で有益な情報となる。また、
本研究で蓄積したリケッチア遺伝子配列情報およびリケッチア分離株は、
病原性リケッチアの特異的診断法の確立や哺乳類における病原性の解析に
有用な研究資源となる。
86
ABSTRACT
Rickettsiae are obligate intracellular Gram-negative bacteria that cause
rickettsioses in humans throughout the world. Ticks harbour most of the members
of spotted fever group (SFG) rickettsiae and transmit them to humans and animals.
In Japan, Rickettsia japonica, a causative agent of Japanese spotted fever (JSF), was
firstly identified as a human pathogen. Several other SFG rickettsiae have also been
reported from both animals and ticks; however, there is scanty information on their
pathogenic potential and the overall diversity of Rickettsia species circulating in
Japan. This thesis aimed to characterise a wide range of SFG rickettsiae in vector
ticks in Japan by analysing multiple rickettsial genes which enables the detailed
phylogenetic classification of SFG rickettsiae and to isolate Rickettsia using
arthropod cell lines.
In chapter I, a nationwide cross-sectional survey was conducted on questing
ticks to understand the overall diversity of SFG rickettsiae in Japan. Out of 2,189
individuals (19 tick species in 4 genera), 373 (17.0%) samples were positive for
Rickettsia spp. by gltA real-time PCR. Conventional PCR and sequencing analyses
of gltA indicated the presence of 15 different genotypes of SFG rickettsiae. Based
on the multiple gene sequence analysis, five Rickettsia species, namely R. asiatica,
R. helvetica, R. monacensis (formerly reported as Rickettsia sp. In56 in Japan), R.
87
tamurae, and Candidatus R. tarasevichiae, and several unclassified SFG rickettsiae
were identified. A strong association between rickettsial genotypes and their host
tick species was observed, while there was little association between rickettsial
genotypes and their geographical origins. These observations may indicate that
most of the SFG rickettsiae have a limited host range and are maintained in certain
ticks in the natural environment.
In chapter II, two arthropod cell lines (ISE6 derived from Ixodes scapularis
tick and C6/36 derived from Aedes albopictus mosquito) were used to isolate
microorganisms from questing ticks. A total of 170 tick homogenates were
inoculated into each cell line. Bacterial growth was confirmed by PCR amplifying
16S ribosomal DNA (rDNA) of eubacteria. During the 16 weeks of observation
period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36
cells, respectively. These included 4 previously validated rickettsial species namely
R. asiatica, R. helvetica, R. monacensis, and R. tamurae and one uncharacterised
rickettsial genotype Rickettsia sp. LON and two tick symbionts, Spiroplasma sp.
and Rickettsiella sp. The use of arthropod cell lines seems promising to expand the
knowledge on microorganisms in ticks.
In conclusion, the present study highlights the wide distribution and high
frequency of SFG rickettsiae in ixodid ticks and provides basic information
88
essential to understand epidemiology of rickettsiosis in Japan. The genetic
information obtained from this study is useful for future development of diagnostic
methods for Rickettsia infections. The bacterial isolates are important to further
analyse their pathogenic potential in vertebrate animals and their roles as symbionts
in ticks.
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